Limits...
Rab14 regulates maturation of macrophage phagosomes containing the fungal pathogen Candida albicans and outcome of the host-pathogen interaction.

Okai B, Lyall N, Gow NA, Bain JM, Erwig LP - Infect. Immun. (2015)

Bottom Line: Interference with endogenous Rab14 did not affect the migration of macrophages toward C. albicans cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing.This was associated with a significant increase in the level of macrophage killing by C. albicans.Therefore, Rab14 activity promotes phagosome maturation during C. albicans infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.

View Article: PubMed Central - PubMed

Affiliation: Aberdeen Fungal Group, University of Aberdeen, Aberdeen, United Kingdom.

Show MeSH

Related in: MedlinePlus

The localization of eGFP-Rab5 to phagosomes containing C. albicans cells is unaffected in Rab14-depleted macrophages. (A) Time taken for Rab5 to be recruited to phagosomes following the engulfment of live C. albicans cells by siRNA-transfected RAW 264.7 macrophages. (B) Duration of Rab5 recruitment to RAW 264.7 macrophages. (A and B) Symbols indicate phagosomes analyzed from at least three independent experiments, with means shown. ANOVA and Bonferroni post hoc tests were carried out. ns, no significant difference. (C and D) J774.1 macrophages transfected with siRNA to knock down Rab14 were lysed, and the protein expression levels of Rab2 (C) and Rab4 (D) were determined by Western blot analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4363425&req=5

Figure 6: The localization of eGFP-Rab5 to phagosomes containing C. albicans cells is unaffected in Rab14-depleted macrophages. (A) Time taken for Rab5 to be recruited to phagosomes following the engulfment of live C. albicans cells by siRNA-transfected RAW 264.7 macrophages. (B) Duration of Rab5 recruitment to RAW 264.7 macrophages. (A and B) Symbols indicate phagosomes analyzed from at least three independent experiments, with means shown. ANOVA and Bonferroni post hoc tests were carried out. ns, no significant difference. (C and D) J774.1 macrophages transfected with siRNA to knock down Rab14 were lysed, and the protein expression levels of Rab2 (C) and Rab4 (D) were determined by Western blot analysis.

Mentions: Rab14 associates with phagosomes transiently, and we hypothesize that this association may be important in the regulation of phagolysosome biogenesis. To further understand the role of Rab14, we investigated whether markers of early and late stages of the phagosome maturation process were affected in macrophages with reduced Rab14 expression. RAW 264.7 macrophages depleted of Rab14 (using siRNA) were cotransfected with eGFP-Rab5 and were infected with live C. albicans cells. Western blot and quantitative PCR (qPCR) analyses were performed to confirm that Rab14 knockdown was maintained in the context of eGFP-Rab5 expression (see Fig. S4 in the supplemental material). Live imaging revealed that Rab5 associated transiently with phagosomes containing C. albicans cells as early as <1 min after uptake and that depletion of Rab14 did not affect the kinetics of this recruitment (Fig. 6A). Rab5 dissociated from phagosomes after 3 min, on average, in control and Rab14 siRNA-treated macrophages (Fig. 6B), suggesting that Rab5 localization to phagosomes containing C. albicans cells occurs prior to Rab14 localization and that the retention kinetics of Rab5 is independent of Rab14.


Rab14 regulates maturation of macrophage phagosomes containing the fungal pathogen Candida albicans and outcome of the host-pathogen interaction.

Okai B, Lyall N, Gow NA, Bain JM, Erwig LP - Infect. Immun. (2015)

The localization of eGFP-Rab5 to phagosomes containing C. albicans cells is unaffected in Rab14-depleted macrophages. (A) Time taken for Rab5 to be recruited to phagosomes following the engulfment of live C. albicans cells by siRNA-transfected RAW 264.7 macrophages. (B) Duration of Rab5 recruitment to RAW 264.7 macrophages. (A and B) Symbols indicate phagosomes analyzed from at least three independent experiments, with means shown. ANOVA and Bonferroni post hoc tests were carried out. ns, no significant difference. (C and D) J774.1 macrophages transfected with siRNA to knock down Rab14 were lysed, and the protein expression levels of Rab2 (C) and Rab4 (D) were determined by Western blot analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4363425&req=5

Figure 6: The localization of eGFP-Rab5 to phagosomes containing C. albicans cells is unaffected in Rab14-depleted macrophages. (A) Time taken for Rab5 to be recruited to phagosomes following the engulfment of live C. albicans cells by siRNA-transfected RAW 264.7 macrophages. (B) Duration of Rab5 recruitment to RAW 264.7 macrophages. (A and B) Symbols indicate phagosomes analyzed from at least three independent experiments, with means shown. ANOVA and Bonferroni post hoc tests were carried out. ns, no significant difference. (C and D) J774.1 macrophages transfected with siRNA to knock down Rab14 were lysed, and the protein expression levels of Rab2 (C) and Rab4 (D) were determined by Western blot analysis.
Mentions: Rab14 associates with phagosomes transiently, and we hypothesize that this association may be important in the regulation of phagolysosome biogenesis. To further understand the role of Rab14, we investigated whether markers of early and late stages of the phagosome maturation process were affected in macrophages with reduced Rab14 expression. RAW 264.7 macrophages depleted of Rab14 (using siRNA) were cotransfected with eGFP-Rab5 and were infected with live C. albicans cells. Western blot and quantitative PCR (qPCR) analyses were performed to confirm that Rab14 knockdown was maintained in the context of eGFP-Rab5 expression (see Fig. S4 in the supplemental material). Live imaging revealed that Rab5 associated transiently with phagosomes containing C. albicans cells as early as <1 min after uptake and that depletion of Rab14 did not affect the kinetics of this recruitment (Fig. 6A). Rab5 dissociated from phagosomes after 3 min, on average, in control and Rab14 siRNA-treated macrophages (Fig. 6B), suggesting that Rab5 localization to phagosomes containing C. albicans cells occurs prior to Rab14 localization and that the retention kinetics of Rab5 is independent of Rab14.

Bottom Line: Interference with endogenous Rab14 did not affect the migration of macrophages toward C. albicans cells, the rate of engulfment, the overall uptake of fungal cells, or early phagosome processing.This was associated with a significant increase in the level of macrophage killing by C. albicans.Therefore, Rab14 activity promotes phagosome maturation during C. albicans infection but is dysregulated on the phagosome in the presence of the invasive hyphal form, which favors fungal survival and escape.

View Article: PubMed Central - PubMed

Affiliation: Aberdeen Fungal Group, University of Aberdeen, Aberdeen, United Kingdom.

Show MeSH
Related in: MedlinePlus